Expression In Breast Carcinoma Research Articles

Hormone Receptor Expression in Invasive Breast Cancer Among Korean Women and Comparison of 3 Antiestrogen Receptor Antibodies

Estrogen receptor (ER) and progesterone receptor (PR) are prognostic markers of breast cancer and predictive markers of response to endocrine therapy. To determine rates of ER and PR expression in invasive breast carcinoma among Korean women, the Breast Pathology Study Group of the Korean Society of Pathologists collected 1198 specimens of invasive breast carcinoma from 6 university hospitals. Immunohistochemical analysis was carried out using 1 antibody against PR and 3 antibodies against ER (1D5, 6F11, and SP1). Specimens were evaluated using the semiquantitative Allred score (scores >2 were considered positive). A total of 1077 cases were interpretable for all 3 anti-ER antibodies. ER expression was positive in 68.5% of cases using SP1, in 59.6% using 1D5, and in 58.9% using 6F11. Of 1073 interpretable cases, PR expression was positive in 51.7% of cases. The frequency distribution of Allred scores revealed a bimodal pattern (complete absence of staining or staining in most cells) for both ER and PR. Patients with discordant results for 2 different ER antibodies showed a median overall survival (between that of double-positive cancer and that of double-negative cancer). Our results showed that the rate of hormone receptor expression in breast carcinomas among Korean patients did not differ from that of western patients. In addition, SP1 was the most sensitive antibody for identifying ER expression in tumors. However, further evaluation is needed to determine which antibody is the best for selecting patients with discordant results who are likely to respond to endocrine therapy.

Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein) in human breast cancer is correlated with favourable prognosis

BackgroundPlasminogen activator inhibitor 1 (PAI-1) overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level.MethodsUsing semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25), and in matched pairs of normal (n = 7) and cancerous breast tissues (n = 7). SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs), an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2) and malignant (n = 6) mammary cell lines as well as breast carcinoma lysates (n = 16) were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10) and cancerous (n = 10) breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression.ResultsSERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008) between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09) towards favourable prognosis when SERBP1 was overexpressed in breast cancer.ConclusionsThe RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a novel breast tumour marker with prognostic significance. Its potential involvement in the plasminogen activator protease cascade warrants further investigation.

Evaluation of UDP-glucuronosyltransferase 2B17 (UGT2B17) and dihydrofolate reductase (DHFR) genes deletion and the expression level of NGX6 mRNA in breast cancer

The present study was aimed to investigate the possible association between 19-base pair (bp) deletion polymorphism of the DHFR gene (rs70991108), null genotype of UDP-glucuronosyltransferase 2B17 (UGT2B17) as well as the expression level of nasopharyngeal carcinoma-associated gene 6 (NGX6) with the risk of breast cancer. This case-control study was done on 236 patients with breast cancer and 203 cancer free women. Detection of 19-bp del of DHFR was done using bi-directional PCR allele-specific amplification and UGT2B17 genotyping was performed using multiplex PCR assay. NGX6 mRNA expression level was determined by quantitative reverse transcriptase PCR in 62 breast cancerous and 62 adjacent non-cancerous tissues. Our finding showed an association between null genotype of UGT2B17 and risk of breast cancer and the null genotype increased susceptibility to breast cancer (OR: 2.99; 95 % CI: 1.94-4.60; p < 0.0001). However, no statistically significant difference was found between breast cancer patients and cancer free normal women regarding 19-bp ins/del of DHFR (χ(2) = 0.91, p = 0.63). Real-time PCR data showed that the relative expression level of NGX6 mRNA was significantly lower in cancerous than that in non-cancerous breast tissue specimens (0.936 ± 0.042 and 1.042 ± 0.039, respectively). However, NGX6 mRNA expression was not correlated with tumors grade (p > 0.05). In conclusion, the null genotype of UGT2B17 revealed to be a risk factor for breast cancer in a sample of Iranian population. Furthermore, down-regulation of NGX6 mRNA expression in breast carcinoma confirms the growing proof regarding the tumor suppressor role of NGX6.

Immunohistochemical analysis of pathway HER2 4B5 performance in breast carcinoma expressing HER4.

16 Background: The ability of pharmacodiagnostic antibodies to specifically detect their target antigen is essential to their performance. Non-clinical protein based models for specificity of two human epidermal growth factor 2 (HER2) pharmacodiagnostic antibodies, Oracle CB11 and PATHWAY HER2 4B5 (4B5) (Ventana Medical Systems Inc., Tucson, AZ, USA), have been reported to demonstrate affinity to both HER2 and HER4. To date there has been no evidence showing that affinity of 4B5 for HER4 impacts the IHC clinical HER2 assay. We therefore sought to investigate whether there exists any risk of the 4B5 antibody detecting HER4-positive but HER2-negative cases as falsely positive due to a hypothetical cross reactivity to HER4. Methods: A HER4 IHC assay was developed by screening ten HER4 antibodies for specificity by staining FFPE blocks of recombinant HEK-293 cells expressing HER1, HER2, HER3 and HER4. Specific HER4 antibodies were then evaluated in breast carcinoma to identify a clone that provided distinct membrane staining in tumour cells. This pattern was used as a reference in order to detect any potential interference of HER4 detection in determining the HER2 score. The HER4 antibody E200 (Epitomics) was selected and used to evaluate a cohort of breast carcinoma cases. The HER2 expression in these cases was then assessed using anti-HER2 clone PATHWAY HER2 4B5 and a HER2 reference antibody, clone SP3 (Spring Bioscience). Results: 117 out of 241 (48%) breast tissue cases evaluated were HER4 positive for membrane staining by IHC using E200. The scoring criteria for determining the HER4 score was based on the system used to score HER2, however a positive score indicated any membrane staining and therefore a 1+, 2+ or 3+ score was considered HER4-positive. 4B5 exhibited similar staining patterns as SP3 in co-expressing HER4 and HER2 tissues indicating that 4B5 was detecting only the HER2 antigen. Eighteen cases were HER4-positive by E200 and level 0 by 4B5 including a HER4 level 3+ case. Conclusions: PATHWAY HER2 4B5, a clinically relevant pharmacodiagnostic antibody for detecting HER2 expression in breast carcinoma, does not demonstrate cross reactivity to HER4 in IHC of FFPE clinical tissues.

227. Maspin expression in breast carcinoma – Relation to established prognostic markers and angiogenesis

227. Maspin expression in breast carcinoma – Relation to established prognostic markers and angiogenesis

An induction of microRNA, miR-7 through estrogen treatment in breast carcinoma

BackgroundEstrogen plays an important role in the development of estrogen-dependent breast carcinoma. Recently, several studies demonstrated a possible involvement of several micro RNAs (miRNAs) in the development of resistance to endocrine therapy in breast cancer patients, but the correlation between estrogen actions and miRNA expression in breast carcinoma still remains largely unknown. Therefore, in this study, we examined the in vitro effects of estrogen upon miRNA expression profiles in breast carcinoma.MethodsWe first screened the miRNA expression profiles induced by 17β-Estradiol (E2) using RT2 miRNA PCR Array in the ER-positive breast carcinoma cell line MCF-7. We identified miR-7 as the important miRNA associated with estrogen actions in these cells and further examined the changes of estrogen-dependent EGFR expression by miR-7 in ER-positive or -negative breast carcinoma cell lines including MCF-7. We also evaluated the correlation between miR-7 and EGFR expression in breast carcinoma cells derived from 21 patients using laser capture microdissection combined with quantitative reverse transcriptase-PCR.ResultsSeventeen miRNAs were significantly induced by E2 treatment in the MCF-7 cell line. Among 17 miRNAs induced by estradiol treatment, only miR-7 expression was significantly decreased by subsequent ICI treatment. The expression of miR-7 was up-regulated 2.94-fold by E2 treatment. miR-7 was reported to suppress epidermal growth factor receptor (EGFR) expression in several human malignancies. Transfection of miR-7 significantly suppressed EGFR mRNA levels in MCF-7 cells. Depletion of E2 from cell culture media also increased the expression level of EGFR mRNA in MCF-7 and T-47D cells but not in ER-negative, MDA-MB-231 and SK-BR-3 cells. We also evaluated the status of miR-7 in breast carcinoma tissues, but the correlation between the status of miR-7 and EGFR in carcinoma cells isolated by laser capture microscopy was not detected.ConclusionsThese results suggest that miR-7 may play a role in the development of resistance to endocrine therapy in breast cancer patients through regulating EGFR expression of carcinoma cells.

Expression of the RET Proto-oncogene Is Regulated by TFAP2C in Breast Cancer Independent of the Estrogen Receptor

The RET proto-oncogene is expressed as part of the estrogen receptor (ER) cluster in breast cancer. We sought to determine if TFAP2C regulates Ret expression directly or indirectly through ER. Chromatin immunoprecipitation sequencing (ChIP-Seq) and gel-shift assay were used to identify TFAP2C binding sites in the RET promoter in four breast cancer cell lines. Ret mRNA and protein levels were evaluated in ER-positive and ER-negative breast cancer cell lines after knockdown of TFAP2C. Luciferase expression assay was performed to assess expression from two of the identified sites. ChIP-Seq identified five main binding peaks for TFAP2C in the RET promoter at -101.5 kb, -50.7 kb, -32.5 kb, +5.0 kb, and +33.6 from the RET transcriptional start site. Binding at three of the AP-2 sites was conserved across all four cell lines, whereas the RET -101.5 and RET +33.6 sites were each found to be unbound by TFAP2C in one cell line. A TFAP2C consensus element was confirmed for all five sites. Knockdown of TFAP2C by siRNA in ER-positive MCF-7 cells resulted in significant down regulation of Ret mRNA compared to nontargeting (NT) siRNA (0.09 vs. 1.0, P < 0.001). Knockdown of TFAP2C in ER-negative MDA-MB-453 cells also led to a significant reduction in Ret mRNA compared to NT siRNA (0.16 vs. 1.0, P < 0.001). In MCF-7 cells, knockdown of TFAP2C abrogated Ret protein expression (0.02 vs. 1.0, P < 0.001) before reduction in ER. TFAP2C regulates expression of the RET proto-oncogene through five AP-2 regulatory sites in the RET promoter. Regulation of Ret by TFAP2C occurs independently of ER expression in breast carcinoma.

Quantitative Proteomics Profiling of Murine Mammary Gland Cells Unravels Impact of Annexin-1 on DNA Damage Response, Cell Adhesion, and Migration

Annexin 1 (ANXA1), the first characterized member of the annexin superfamily, is known to bind or annex to cellular membranes in a calcium-dependent manner. Besides mediating inflammation, ANXA1 has also been reported to be involved in important physiopathological implications including cell proliferation, differentiation, apoptosis, cancer, and metastasis. However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear. To elucidate how ANXA1 plays a role in breast cancer initiation, we performed stable isotope labeling of amino acids in cell culture analysis on normal mammary gland epithelial cells from ANXA1-heterozygous (ANXA1(+/-)) and ANXA1-null (ANXA1(-/-)) mice. Among over 4000 quantified proteins, we observed 214 up-regulated and 169 down-regulated with ANXA1(-/-). Bioinformatics analysis of the down-regulated proteins revealed that ANXA1 is potentially implicated in DNA damage response, whereas the analysis of up-regulated proteins showed the possible roles of ANXA1 in cell adhesion and migration pathways. These observations were supported by relevant functional assays. The assays for DNA damage response demonstrated an accumulation of more DNA damage with slower recovery on heat stress and an impaired oxidative damage response in ANXA1(-/-) cells in comparison with ANXA1(+/-) cells. Overexpressing Yes-associated protein 1 or Yap1, the most down-regulated protein in DNA damage response pathway cluster, rescued the proliferative response in ANXA1(-/-) cells exposed to oxidative damage. Both migration and wound healing assays showed that ANXA1(+/-) cells possess higher motility with better wound closure capability than ANXA1(-/-) cells. Knocking down of β-parvin, the protein with the highest fold change in the cell adhesion protein cluster, indicated an increased cell migration in ANXA1(-/-) cells. Altogether our quantitative proteomics study on ANXA1 suggests that ANXA1 plays a protective role in DNA damage and modulates cell adhesion and motility, indicating its potential role in cancer initiation as well as progression in breast carcinoma.

Automated Quantitative Analysis of p53, Cyclin D1, Ki67 and pERK Expression in Breast Carcinoma Does Not Differ from Expert Pathologist Scoring and Correlates with Clinico-Pathological Characteristics

There is critical need for improved biomarker assessment platforms which integrate traditional pathological parameters (TNM stage, grade and ER/PR/HER2 status) with molecular profiling, to better define prognostic subgroups or systemic treatment response. One roadblock is the lack of semi-quantitative methods which reliably measure biomarker expression. Our study assesses reliability of automated immunohistochemistry (IHC) scoring compared to manual scoring of five selected biomarkers in a tissue microarray (TMA) of 63 human breast cancer cases, and correlates these markers with clinico-pathological data. TMA slides were scanned into an Ariol Imaging System, and histologic (H) scores (% positive tumor area x staining intensity 0–3) were calculated using trained algorithms. H scores for all five biomarkers concurred with pathologists’ scores, based on Pearson correlation coefficients (0.80–0.90) for continuous data and Kappa statistics (0.55–0.92) for positive vs. negative stain. Using continuous data, significant association of pERK expression with absence of LVI (p = 0.005) and lymph node negativity (p = 0.002) was observed. p53 over-expression, characteristic of dysfunctional p53 in cancer, and Ki67 were associated with high grade (p = 0.032 and 0.0007, respectively). Cyclin D1 correlated inversely with ER/PR/HER2-ve (triple negative) tumors (p = 0.0002). Thus automated quantitation of immunostaining concurs with pathologists’ scoring, and provides meaningful associations with clinico-pathological data.

Maspin expression in breast carcinoma

Maspin expression in breast carcinoma

HER2 Expression in Breast Cancer With Nonamplified HER2 and Gains of Chromosome 17 Centromere

Gains of chromosome 17 centromere (CEP17) may be accompanied by gains of chromosome 17q. To evaluate the effect of CEP17 gains (CEP17>3 copies per tumor nucleus) on the expression of the HER2 gene, which is located on chromosome 17q12-21.32, we analyzed HER2 amplification and expression in breast carcinomas with and without CEP17 gains. We isolated tumor nuclei from frozen tissues of 37 breast carcinomas for analysis of the HER2 gene and CEP17 by fluorescence in situ hybridization. HER2 expression was detected by immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded sections of the corresponding tumors. Tumors with amplified HER2 as determined by both HER2 copy number and HER2/CEP17 ratio were detected in 29.7% (11/37). CEP17 gains were significantly associated with HER2 amplification (P=0.005) but not associated with estrogen receptor status, tumor grade, and lymph node status (P>0.05). In contrast, HER2 amplification was significantly associated with estrogen receptor negativity (P=0.020) but not with tumor grade and lymph node status (P>0.05). IHC analysis was performed in 7 HER2-amplified tumors and all of these were IHC 3+, which were used as positive controls. Among HER2-non-amplified tumors with CEP17 gains, only 1 tumor (1/8, 12.5%) was IHC 3+. However, none of the HER2-non-amplified tumors without CEP17 gains was IHC 3+. In HER2-non-amplified tumors, there was no significant association between HER2 protein expression as detected by IHC and CEP17 or HER2 copy number (P=0.999, P=0.785, respectively). These findings indicate that in the absence of HER2 amplification, CEP17 gains do not have a significant effect on HER2 protein expression.

Suppressor of Cytokine Signaling 3 Inhibits Breast Tumor Kinase Activation of STAT3

Breast tumor kinase (Brk) was originally isolated from a human metastatic breast tumor, but also is found expressed in other epithelial tumors and in a subset of normal epithelia. Brk is a tyrosine kinase and its expression in breast carcinoma has been linked to tumor progression. The signal transducer and activator of transcription 3 (STAT3) is one of the substrate targets of Brk, and elevated tyrosine phosphorylation of STAT3 is known to contribute to oncogenesis. Conventional activation of STAT3 occurs in response to cytokine stimulation of Janus tyrosine kinases (JAK). One of the negative regulators discovered in cytokine signaling of the JAK-STAT pathway is the suppressor of cytokine signaling 3 (SOCS3). In this report we describe the finding that SOCS3 can also inhibit the unconventional target, Brk. Investigation of the mechanism by which SOCS3 inhibits Brk reveals the SOCS3 protein binds to Brk primarily via its SH2 domain, and its main inhibitory effect is mediated by the SOCS3 kinase inhibitory region (KIR). SOCS3 has only a modest effect on promoting Brk degradation, and this requires the C-terminal SOCS box domain. SOCS3 is the only known inhibitor of Brk, and knowledge of the mechanisms by which SOCS3 inhibits Brk may lead to methods that block Brk in cancer progression.

33P C-Kit Expression in Breast Carcinomas: Correlation with other Clinico-Pathological Parameters

ABSTRACT Background c-KIT is a transmembrane tyrosine kinase representing a relevant target for specific therapy in some malignancies. The expression and function of c-KIT in breast cancer is quite controversial subject. Method A total of 117 consecutive cases of invasive ductal breast carcinomas diagnosed in our institution were evaluated for c-KIT immunoexpression. The findings were correlated with patient and tumor characteristics. Results Thirteen (11%) cases expressed c-KIT. The c-KIT positive cancers were different from c-KIT negative carcinomas by having a high histological grade (46% vs. 18%, p = 0.062), tumor size larger than 2cm (61% vs. 33%, p = 0.041), pushing border (38% vs. 13%, p = 0.035), and by marked tumor necrosis (31% vs. 5%, p = 0.001). Triple-negative phenotype showed 6 (46%) cases from the group of c-KIT positive tumors (p = 0.003). No significant differences were found between c-KIT positive and c-KIT negative cancer group in relation to patient’s age and lymphoid infiltration. Conclusion c-KIT expression is associated with poor prognostic tumor characteristics. These data suggest that c-KIT expression might have some clinical and therapeutic implications in breast cancer. Disclosure All authors have declared no conflicts of interest.

96P Quantitative Proteomics Profiling of Murine Mammary Gland Cells Unravels Impact of Annexin-1 On Dna Damage, Cell Adhesion and Migration

ABSTRACT Modern mass spectrometry (MS)-based technology offers exciting strategies for advances in many areas of biology. Among the quantitative MS strategies, stable isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and robust approach to solve a wide range of biological problems. We employ this technology to understand the roles of Annexin-1 (ANXA1) that has been reported to be involved in important physio-pathological implications including cell proliferation, differentiation, apoptosis, cancer and metastasis. However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear. In this study, we perform quantitative comparison of the proteomes of normal mammary gland cells from ANXA1 heterozygous (ANXA1+/-) and null (ANXA1-/-) mice using SILAC. Among over 4000 quantified proteins, we observe 214 up-regulated and 169 down-regulated in ANXA1-/-. Bioinformatics analysis of the up-regulated proteins reveals that ANXA1 is potentially implicated in DNA damage and apoptosis, whereas the analysis of down-regulated proteins shows ANXA1's possible roles in cell adhesion and migration pathways. Results of relevant functional assays further support the bioinformatics analysis. Among them, the Comet assay demonstrates that ANXA1-/- cells accumulates more DNA damage with slower recovery as compared to the ANXA1+/- after heat stress. Both migration and wound healing assays show that ANXA1+/- cells possess higher motility with better wound closure capability than the ANXA1-/- . Our quantitative proteomics data together with functional analysis strongly suggest that ANXA1 plays a protective role in DNA damage and modulates cell adhesion and motility, demonstrating its potential role in cancer initiation as well as progression in breast carcinoma. Disclosure All authors have declared no conflicts of interest.

Clinical significance of CC3/TIP30 expression in breast carcinoma and its correlation with HER-2/neu

To explore the clinical significance of CC3/TIP30 protein's expression in breast carcinoma and its correlation with HER-2/neu. The expression of CC3/TIP30 and HER-2/neu protein was detected in 112 breast cancer tissues which was collected from January 2004 to January 2005 by immunohistochemistry and the relationship with clinic pathological parameters and prognosis was analyzed. Small interfering RNA (siRNA) which target to knock out CC3/TIP30 were transfected into SK-BR-3 cells. Real-time PCR were used to detect the level of CC3/TIP30 and HER-2/neu mRNA. The results of immunohistochemistry showed CC3/TIP30 protein was correlated with TNM stage, lymph node status, HER-2 status and molecule classification (P = 0.048, 0.019, 0.027, 0.011), but there was no association with age, tumor size, estrogen receptor and progesterone receptor. Real-time PCR results revealed that CC3/TIP30 siRNA down-regulation the level of its mRNA, accompanied by a decline in the expression of HER-2/neu gene mRNA, the difference was statistically significant (F = 56.797, P = 0.000; F = 165.101, P = 0.000). In addition, Kaplan-Meier curves of disease-specific survival analysis showed a marked difference in the subtype of HER-2 protein positive between CC3/TIP30 positive group and negative group (χ(2) = 10.732, P = 0.001). The loss of CC3/TIP30 is related to occurrence and development in breast cancer, suggesting early onset of metastasis and recurrence. Perhaps CC3/TIP30 can be considered as a sub-typing indicator in HER-2 positive breast cancer.

The Prognostic Value of Breast Cancer Resistance Protein (BCRB/ABCG2) Expression in Breast Carcinomas

In cancer, the level of expression of breast cancer resistance protein (BCRP/ABCG2) can vary depending on cell type. However, its precise role in breast cancer has been controversial. The aim of this study was to investigate the expression of ABCG2 in breast carcinomas and relate the results to the established prognostic factors. An immunohistochemical study was conducted on 200 breast carcinoma specimens using the avidin-biotin peroxidase method. ABCG2 was expressed in 77% of cases of invasive ductal carcinoma. There was a statistically significant correlation between ABCG2 expression and each of the tumor grade, clinical stage, lymph node metastasis, and HER2 immunostaining, but no such association with progesterone receptor (PR) and estrogen receptor (ER) status. Increased expression of ABCG2 in invasive ductal carcinoma cells and its statistically significant correlation with HER2 expression are strongly correlated with tumor progression, invasion, and metastasis in human breast cancer and indicates that ABCG2 can be used as a target for the development of novel therapies.

Correlation of hormone receptors with her-2 neu protein expression and the histological grade in invasive breast cancers in a cohort of saudi arabia

Data on Hormone Receptor and Her-2/neu expression in breast cancers from Saudi Arabia and Gulf Region is sparse. We undertook this study to describe the patterns of hormone receptor and Her-2/neu protein expression in breast carcinoma and compared them with the histological grade at a University Hospital in Riyadh. We conducted a retrospective study on 164 invasive ductal carcinoma of breast between the year 2002 and 2008. Immunohistochemical analysis for Estrogen and Progesterone Receptor and Her-2/neu was done in all the cases. Fluorescent in situ hybridization for Her-2/neu gene amplification was performed in all 2+ cases and few equivocal 1+ and 3+cases by immunohistochemistry. Correlation between Estrogen and Progesterone Receptor and Her-2/ neu amplification and grade of tumor was calculated. The expression of Estrogen Receptor, Progesterone Receptor were significantly correlated (p < 0.001). Also, there was a significant negative correlation between expression of hormone receptor and Her-2/neu amplification. The histologic grade of the tumor was significantly correlated to the expression of both Estrogen and Progesterone Receptor. However, the relationship between Her-2/ neu amplification and grade was not significant and many of the grade III tumor were Her-2/neu negative. In addition, Her-2/neu gene amplification by fluorescent in situ hybridization was observed in 84.6% of breast cancer that were 3+ and in 18.75 % cases that were 2+ by immunohistochemistry. Prevalence of Estrogen and Progesterone Receptor expression and Her-2/neu amplification in breast cancer in the Saudi Arabian population is similar to that reported internationally. There is a negative correlation between hormone receptors expression and Her-2/neu amplification. However not all of the high-grade breast cancers showed Her-2/neu positive status.

P1-06-07: TIMP-1 in Combination with HER2 and TOP2A for Prediction of Benefit from Adjuvant Anthracyclines in High Risk Breast Cancer Patients.

Abstract Purpose: HER2 amplification, TOP2A aberrations and absence of TIMP-1 (Tissue Inhibitor of Metalloproteinase-1) expression in breast carcinomas have been associated with incremental benefit from anthracycline-containing adjuvant chemotherapy in several reports. In the DBCG 89D trial, we demonstrated that the predictive value of these markers improved when they were combined in a profile and the present study was undertaken to validate these findings in NCIC CTG MA.5, a similar but independent clinical trial. Design: TIMP-1 was examined by immunohistochemistry in archival tumor tissue from 403 of 716 premenopausal high-risk patients with known HER2 and TOP2A status who were randomized to CEF or CMF in the MA.5 trial. Patients were classified according to 2 predefined marker profiles — the HT profile (HER2, TIMP-1) and the 2T profile (TOP2A, TIMP-1) and the statistical analyses were performed as closely as possible to the analytical approach used previously in the MA.5 trial and when analysing the biomarker profiles in the DBCG 89D trial. Results: 98 (24%) patients had no TIMP-1 staining of tumor cells, 27% were HER2 amplified, and 18% were TOP2A aberrant. 44% of patients were classified as HT responsive (HER2-positive and/or TIMP-1 negative) and 37% as 2T responsive (TOP2A aberrant and/or TIMP-1 negative). There was no heterogeneity in treatment effect of CEF versus CMF according to TIMP-1. In HT responsive patients, CEF was superior to CMF with improved RFS (adjusted HR, 0.64; 95% CI, 0.42 to 0.98) and a borderline-significant improvement in OS (adjusted HR, 0.66; 95% CI, 0.42 to 1.04). A significant HT profile versus treatment interaction was detected for OS (P=0.03). In 2T responsive patients, CEF was superior to CMF with borderline significant improvement in RFS (adjusted HR, 0.67; 95% CI, 0.43 to 1.03), and with improvement in OS (adjusted HR, 0.58; 95% CI, 0.36 to 0.93). A significant 2T profile versus treatment interaction was detected for OS (P=0.01). Conclusion: In the MA.5 trial, we have validated the HT and 2T profiles as predictors of incremental benefit from anthracycline-containing chemotherapy. The proportion of patients categorized as anthracycline responsive increases from 18–27% using individual markers to 37–44% when combining TIMP-1 with either HER2 or TOP2A. Patients with responsive profiles had a 34–42% relative reduction in mortality when treated with CEF. In contrast, patients with non-responsive profiles (56-63% of patients) had no incremental benefit from CEF compared with CMF. All 3 biomarkers are easily applied in the pathology lab and as such could be used in daily clinical practice to select patients for anthracycline or non-anthracycline containing adjuvant chemotherapy. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-07.

TIMP-1 in combination with HER2 and TOP2A for prediction of benefit from adjuvant anthracyclines in high-risk breast cancer patients

HER2 amplification, TOP2A aberrations, and absence of tissue inhibitor of metalloproteinase (TIMP-1) expression in breast carcinomas have been shown to be associated with incremental benefit from anthracycline-containing adjuvant chemotherapy, and this study was undertaken to validate these findings in a similar, but independent, randomized clinical trial. TIMP-1 was examined by immunohistochemistry in archival tumor tissue from 403 of 716 premenopausal high-risk patients with known HER2 and TOP2A status who were randomized to cyclophosphamide, epirubicin, and fluorouracil (CEF) or cyclophosphamide, methotrexate, and fluorouracil (CMF) in the MA.5 trial. Ninety-eight (24%) patients had no TIMP-1 staining of tumor cells, 27% were HER2 amplified, and 18% were TOP2A aberrant. Forty-four percentage was classified as HT responsive (HER2 amplified and/or TIMP-1 negative) and 37% as 2T responsive (TOP2A aberrant and/or TIMP-1 negative). There was no heterogeneity in treatment effect of CEF versus CMF according to TIMP-1. In HT-responsive patients, CEF was superior to CMF with an improved RFS (adjusted HR, 0.64; 95% CI, 0.42-0.97), but this was not significant for OS (adjusted HR, 0.66; 95% CI, 0.42-1.04). A significant HT profile versus treatment interaction was detected for OS (P = 0.03). In 2T-responsive patients, CEF seemed to improve RFS compared to CMF (adjusted HR, 0.67; 95% CI, 0.43-1.03) and improved OS (adjusted HR, 0.58; 95% CI, 0.36-0.93). A significant 2T profile versus treatment interaction was detected for OS (P = 0.01). With this study, we validate a more substantial reduction in mortality by CEF compared to CMF in patients with an HT- or 2T-responsive profile; however, we could not show a similarly significant reduction in RFS events, where a benefit of CEF over CMF was found irrespective of TIMP-1 status. Further studies are necessary before the HT and 2T profiles may be used to direct the use of anthracyclines.

Endogenous Myoglobin in Breast Cancer Is Hypoxia-inducible by Alternative Transcription and Functions to Impair Mitochondrial Activity: A ROLE IN TUMOR SUPPRESSION?

Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here, we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/2-dependent transactivation. The hypoxia-induced MB mRNA originated from a novel alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O(2) uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O(2) consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O(2). Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.